Lentiviral vectors useful for the treatment of disease

Abstract

This disclosure relates generally to lentiviral vectors useful for the treatment of a disease or condition, for example, Wiskott-Aldrich Syndrome (WAS) or Sickle Cell Disease (SCD).

What is claimed is: 
     
         1 . A lentiviral vector, comprising:
 a first promoter operably linked to a first nucleic acid sequence, wherein the first nucleic acid sequence encodes a Wiskott-Aldrich Syndrome protein; and   a modified HS4-650 insulator, wherein:   when present in the vector, the modified HS4-650 insulator comprises an inactivated splice acceptor site 1 (SA1) relative to an unmodified HS4-650 insulator, and wherein:
 SA1 is present in an unmodified HS4-650 insulator at nucleotide positions 385-386 with numbering relative to SEQ ID NO:2, wherein SEQ ID NO:2 is the reverse, complement sequence of the unmodified HS4-650 insulator set forth in SEQ ID NO:1; and/or 
 SA1 comprises the sequence TTGCATCCAG{circumflex over ( )}ACACCATCAA (SEQ ID NO:60), where {circumflex over ( )} represents the splice position. 
   
     
     
         2 . The lentiviral vector of  claim 1 , wherein the modified HS4-650 insulator comprises, relative to an unmodified HS4-650 insulator, a mutation that inactivates SA1. 
     
     
         3 . The lentiviral vector of  claim 2 , wherein the mutation is a mutation of the A at position 384 and/or a mutation of the G at position 385, with numbering relative to SEQ ID NO:2. 
     
     
         4 . (canceled) 
     
     
         5 . The lentiviral vector of  claim 1 , wherein the modified HS4-650 insulator comprises the sequence set forth in any one of SEQ ID NOs:3, 12, 21, 30, 39 and 48. 
     
     
         6 . The lentiviral vector of  claim 2 , wherein the modified HS4-650 insulator comprises a mutation that inactivates splice acceptor site 2 (SA2) relative to an unmodified HS4-650 insulator, wherein SA2 is present in an unmodified HS4-650 insulator at nucleotide positions 446-447, with numbering relative to SEQ ID NO:2. 
     
     
         7 . The lentiviral vector of  claim 6 , wherein the mutation is a mutation of the A at position 445 and/or a mutation of the G at position 446, with numbering relative to SEQ ID NO:2. 
     
     
         8 . (canceled) 
     
     
         9 . The lentiviral vector of  claim 6 , wherein the reverse complement sequence of the modified HS4-650 insulator comprises the sequence set forth in any one of SEQ ID NOs:4, 13, 22, 31, 40 and 49. 
     
     
         10 . The lentiviral vector of  claim 2 , wherein the modified HS4-650 insulator comprises a mutation that inactivates splice acceptor site 3 (SA3) relative to an unmodified HS4-650 insulator, wherein SA3 is present in an unmodified HS4-650 insulator at nucleotide positions 456-457, with numbering relative to SEQ ID NO:2. 
     
     
         11 . The lentiviral vector of  claim 10 , wherein the mutation is a mutation of the A at position 455 and/or a mutation of the G at position 456 with numbering relative to SEQ ID NO:2. 
     
     
         12 . (canceled) 
     
     
         13 . The lentiviral vector of  claim 10 , wherein the reverse complement sequence of the modified HS4-650 insulator comprises the sequence set forth in SEQ ID NOs:5, 6, 14, 15, 23, 24, 32, 33, 41, 42, 50 and 51. 
     
     
         14 . The lentiviral vector of  claim 1 , wherein the modified HS4-650 insulator is in the opposite orientation to the first nucleic acid sequence. 
     
     
         15 . The lentiviral vector of  claim 1 , wherein the first nucleic acid is in the forward orientation and the modified HS4-650 insulator is in the reverse orientation within the lentiviral vector. 
     
     
         16 . The lentiviral vector of  claim 1 , wherein the modified HS4-650 insulator is in the same orientation as the first nucleic acid sequence, thereby inactivating SA1. 
     
     
         17 . The lentiviral vector of  claim 16 , wherein the first nucleic acid and the modified HS4-650 insulator are in the forward orientation within the lentiviral vector. 
     
     
         18 . A lentiviral vector, comprising:
 a first promoter operably linked to a first nucleic acid sequence, the first nucleic acid sequence encoding a Wiskott-Aldrich Syndrome protein; and   a modified HS4-650 insulator, wherein:   when present in the vector, the modified HS4-650 insulator comprises an inactivated splice acceptor site 2 (SA2) relative to an unmodified HS4-650 insulator, and wherein:
 SA2 is present in an unmodified HS4-650 insulator at nucleotide positions 446-447, with numbering relative to SEQ ID NO:2, wherein SEQ ID NO:2 is the reverse, complement sequence of the unmodified HS4-650 insulator set forth in SEQ ID NO:1; and/or 
 SA2 comprises the sequence ATCCCCCCAG{circumflex over ( )}GTGTCTGCAG (SEQ ID NO:61), where {circumflex over ( )} represents the splice position. 
   
     
     
         19 - 34 . (canceled) 
     
     
         35 . A lentiviral vector, comprising:
 a first promoter operably linked to a first nucleic acid sequence, the first nucleic acid sequence encoding a Wiskott-Aldrich Syndrome protein; and   a modified HS4-650 insulator, wherein:   when present in the vector, the modified HS4-650 insulator comprises an inactivated splice acceptor site 3 (SA3) relative to an unmodified HS4-650 insulator, and wherein:
 SA3 is present in an unmodified HS4-650 insulator at nucleotide positions 456-457, with numbering relative to SEQ ID NO:2, wherein SEQ ID NO:2 is the reverse, complement sequence of the unmodified HS4-650 insulator set forth in SEQ ID NO:1; and/or 
 SA3 comprises the sequence GTGTCTGCAG{circumflex over ( )}GCTCAAAGAG (SEQ ID NO:62), where {circumflex over ( )} represents the splice position. 
   
     
     
         36 - 51 . (canceled) 
     
     
         52 . The lentiviral vector of  claim 1 , wherein the modified HS4-650 insulator is downstream of the first nucleic acid sequence. 
     
     
         53 . The lentiviral vector of  claim 1 , wherein the Wiskott-Aldrich Syndrome protein comprises an amino acid sequence set forth in SEQ ID NO: 76 or a sequence having at least 95% sequence identity thereto. 
     
     
         54 . (canceled) 
     
     
         55 . The lentiviral vector of  claim 1 , further comprising a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE) between the first nucleic acid sequence and the modified HS4-650 insulator. 
     
     
         56 . (canceled) 
     
     
         57 . The lentiviral vector of  claim 1 , comprising a sequence selected from the group consisting of: the sequence set forth as nucleotides 3098-6006 of SEQ ID NO:57 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; the sequence set forth as nucleotides 3098-6009 of SEQ ID NO:58 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and the sequence set forth as nucleotides 3098-6006 of SEQ ID NO:59 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. 
     
     
         58 . The lentiviral vector of  claim 1 , wherein the first promoter is an MND promoter. 
     
     
         59 . (canceled) 
     
     
         60 . The lentiviral vector of  claim 1 , comprising a sequence selected from the group consisting of: the sequence set forth as nucleotides 2710-6006 of SEQ ID NO:57 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; the sequence set forth as nucleotides 2710-6009 of SEQ ID NO:58 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and the sequence set forth as nucleotides 2710-6006 of SEQ ID NO:59 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. 
     
     
         61 - 69 . (canceled) 
     
     
         70 . The lentiviral vector of  claim 1 , further comprising a polyadenylation signal downstream of the first nucleic acid and the modified HS4-650 insulator. 
     
     
         71 . The lentiviral vector of  claim 1 , that is a plasmid or a viral particle. 
     
     
         72 . (canceled) 
     
     
         73 . (canceled) 
     
     
         74 . A host cell, wherein the host cell comprises the lentiviral vector of  claim 1  or has been transduced with the lentiviral vector of  claim 71 . 
     
     
         75 . (canceled) 
     
     
         76 . The host cell of  claim 74 , wherein the host cell is a hematopoietic stem cell (HSC). 
     
     
         77 - 82 . (canceled) 
     
     
         83 . A method of treating a subject with Wiskott-Aldrich Syndrome, comprising administering to the subject the host cell of  claim 74 . 
     
     
         84 - 87 . (canceled) 
     
     
         88 . The lentiviral vector of  claim 1 , comprising the sequence set forth in SEQ ID NO:84 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.